Note: Descriptions are shown in the official language in which they were submitted.
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A MICROBIOCIDAL FORMULATION
The present invention relates to a microbiocidal formulation
for dissolution in water as a diluent to form a microbiocidal
solution, said formulation containing an alkalinity
neutralising agent to neutralise the total alkalinity present
in the water used as the diluent.
It is well known how to produce a whole range of biocides,
microbiocides and disinfectants from halogen donors,
particularly those of chlorine and iodine.
Typical of such donors are sodium and calcium hypochlorite,
chloramines, iodophors and sodium dichloroisocyanurate
(NaDCC). While the effectiveness of all of these donors as a
source of microbiocidally effective agent, e.g. hypochlorous
acid, has been shown in various laboratory trials at
different concentrations of the agent against a wide range of
micro-organisms, it has been found that, in the field, the
microbiocidal efficiency was not as expected from the
laboratory trials.
After detailed investigation and further tests, it was
surprisingly learnt that the factor most influencing the
effectiveness of the microbiocidal solution was the water
used in its preparation.
The invention provides a microbiocidal formulation suitable,
following dissolution in a diluent to form a microbiocidal
solution for microbiocidal treatment of an environment, the
formulation comprising sufficient diluent alkalinity
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neutralising agent so that, following dissolution in the
diluent, an alkalinity of no more than 100, preferably no
more than 50 mg/1 bicarbonate alkalinity is observed in the
microbiocidal solution; sufficient pH neutralising agent so
that, following dissolution in the diluent, a pH of 5.0-8.0,
preferably 6.0-6.8; is observed in the microbiocidal solution
and a microbiocidally effective amount of an alkalinity
sensitive microbiocidal agent; the formulation being adapted
to release the microbiocidally effective amount of the
alkalinity sensitive microbiocidal agent over a
microbiocidally effective period of time.
Preferably, the alkalinity sensitive microbiocidal agent is a
source of available halogen.
More preferably, the formulation is adapted to release
available halogen, in use, from an organic source/precursor
of hypohalous acid and/or hypohalite.
Advantageously, the organic source/precursor compound
releases, in aqueous solution, hypohalous acid and/or
hypohalite in a microbiocidally effective amount over the
microbiocidally effective period of time, by adaption of the
formulation for the control of the pH.
More advantageously, the alkalinity neutralising agent is
comestibly acceptable.
Preferably, the organic source/precursor compound is a
halogenated isocyanuric compound or a salt thereof.
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More preferably, the source/precursor compound of the
microbiocidally effective hypohalous acid and/or hypohalite
is selected from sodium dihaloisocyanurate, potassium
dihaloisocyanurate or trihaloisocyanuric acid, preferably
sodium dichloroisocyanurate.
Even more preferably, the microbiocidally effective amount of
the hypohalous acid is released from 1 to 5000 ppm of the
solid source/precursor compound and the microbiologically
effective period of time is in the range of 10 seconds to 48
hours.
Advantageously, the micro-organism is selected from E.coli or
Pseudomonas or is a resistant micro-organism, more preferably
a pasteurisation resistant micro-organism, still more
preferably a thermoduric or thermophilic organism, most
preferably, a species of micro-organism selected from
Bacillus, Micrococcus, Microbacterium, Clostridium, Listeria,
AZcaliigenes, Arthrobacter, Lactobacillus, Serratia or any
other spore forming species.
More advantageously, the environment comprises an
external surface or a lumen of an apparatus used in the
production, preparation or processing of food or beverages.
Even more advantageously, the environment comprises process
- liquid or liquid for human or animal consumption.
Preferably, the alkalinity neutralising agent is a comestibly
acceptable acid or its salt or a mixture thereof, preferably
a comestibly acceptable organic acid.
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More preferably, the alkalinity neutralising agent is
succinic acid or a salt thereof.
Even more preferably, the alkalinity neutralising agent is
citric acid or a salt thereof.
Advantageously, the microbiocidal formulation is in powder,
granulate or tablet form.
It is postulated that alkalinity affects the microbiocidal
activity of the hypohalous acid and/or its hypohalite salts
by neutralising the hypohalous acids themselves and by
affecting the pH of the microbiocidal formulation in the
environment.
Upon investigation of the normal or "field" water used as the
diluent, as opposed to the distilled water commonly used in
laboratory trials, bicarbonate alkalinity or total alkalinity
was identified as the cause of the reduced effectiveness of
the microbiocidal solution. This explains why the field
results were disappointing when compared to the laboratory
tests.
It was found that dissolved bicarbonate mineral salts were
combining with the dissociation product of the halogen donor
and reducing its availability for microbiocidal action. This
was particularly noticeable where the microbiocidal solution
had to act at a low (low freely available chlorine
concentration) level in a large volume of diluent water. The
larger the volume/proportion of diluent water compared to the
microbiocide, the more dissolved bicarbonate salts were
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available to combine with the microbiocide agent and to
significantly reduce its effectiveness.
Most surprisingly, this factor does not appear to have been
5 taken into account in the prior art and the applicants are
unable to find prior art on the relative microbiocidal
effectiveness of known microbiocides in field conditions of
varying diluent total alkalinity.
Tests were conducted to compare laboratory trials using
distilled water or water of a known alkalinity as the
diluent. These comparisons provided new and very surprising
results with regard to the efficiency of the microbiocidal
agent when dissolved in high alkalinity water as the diluent.
In order to understand how the total alkalinity of "field"
water could impact on microbiocidal activity, a study was
undertaken to understand what "total alkalinity" or ANC (acid
neutralising capacity) actually was.
Alkalinity is the sum of all titratable bases and is a
measure of the acid neutralising capacity (ANC). Water total
alkalinity is mainly a sum of carbonate, bicarbonate and
hydroxide levels but may also include contributions from
borates, phosphates, silicates or other bases, if these are
present. Bicarbonates are the main contributors to "field"
water total alkalinity.
Alkalinity of water is pH related but is not pH dependant,
for example, water can have a pH of 6.4 and an alkalinity of
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~ ~
w~ ~~ ~~~ ~~ ~~ ~~ ~~ ~~
~ ~ ~ ~ ~~ ~ ~ ~w~ ~ ~ ~ ~
~ 1 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ w w
~ ~ ~ ~ ~ 1
~ 1 ~ ~ 1 ~ ~ ~ ~ ~ ~ ~ 1
~ ~ ~ ~ ~ 1
6
320 mg/1 and, conversely, water can have a pH of 8.0 and an
alkalinity of 95 mg/1. Reference is made to Figure 4 of the
accompanying drawings showing the lack of relationship
between pH and total alkalinity of various field waters
obtained in Ireland.
Geological factors are a major influence on the levels of
total alkalinity likely to be found in water supplies. Thus,
the greater the water residence time in a limestone/dolomite
region, the greater the likelihood of a high alkalinity.
Conversely, water from granite/sandstone areas will have a
low or lower total alkalinity.
Some of the definitions used herein have quite specific
meanings in relation hereto and are now explained.
The expression "ANC" means "acid neutralising capacity".
This term is now coming into use to replace the classical
expression "total alkalinity", although the latter remains
the most widely used term in the field of applied water
chemistry. Outside the field of applied water chemistry,
total alkalinity as a concept separate from pH is not at all
well understood.
The expressions "FAH" and "FAC" mean "freely available
halogen" and "freely available chlorine", respectively, that
is, halogen or chlorine in a microbiocidally active form.
Concerning chlorine, the total concentrations of chlorine,
hypochlorous acid and hypochlorite ion are the "FAC" of the
microbiocidal solution - except at extremely low pH
conditions, the chlorine can be ignored. Figure 1 of the
c~~~ AMENDED SHEET
wJ..
~iZ~.
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accompanying drawings illustrates the pH distribution curves
for each of the three FAC species (it is necessary to specify
a CI concentration of l.OmM and a temperature of 28°C.
Figure 2 shows a simplified pH distribution curves for the
FAC species, based on reasonably ignoring the significant C1z
contribution when the pH is greater than 3 (it is not now
necessary to specify the C12 concentration). Figure 3 shows
the pH dependence of these two FAC species, in which the
respective percentages are on a molar basis.
By example, a chlorine donor compound is often referred to as
having say 500mg of available chlorine per gram. This
describes the total amount of chlorine available but
describes neither the form, its activity, nor its
availability at a given time, all of which will affect the
microbiocidal effectiveness of the solution.
Lubricating agents are commonly used in the tabletting
process e.g. adipic acid or succinic acid. However, their
sole disclosed function to date has been as a lubricating
agent or as the replacement of an existing lubricating agent.
No attempt had been made in the prior art to adapt these
compounds or the like to act as a neutralising agent with
regard to the impact of bicarbonate alkalinity on the
efficacy of the microbiocidal agent in any given
microbiocidal solution.
Many other compounds other than those identified above can be
introduced to perform this function such as, for example, any
comestibly acceptable acid or a salt thereof. However, where
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practical, the level of a suitable lubricating agent should
be adjusted to accommodate or neutralise the impact of the
total alkalinity or ANC value of the diluent water on the
microbiocidal effectiveness of any given microbiocidal
solution.
Cognisant of this, the lubricating component has been taken
into account when calculating the adjustment that would be
required to a "conventional" microbiocidal formulation to
neutralise the total alkalinity or ANC of any given diluent
water. As previously stated, total alkalinity is pH related
but not pH dependant and the use of a comestible acid to
neutralise the alkalinity had the additional benefit of
achieving an optimum pH for the dissociation of the chlorine
donor NaDCC. Thus, a comestible acid (or a salt thereof) was
selected for further tests.
As the object of the present invention was to neutralise
(reduce or eliminate) the ANC of the diluent water, once the
formulation was added to the diluent water and full
dissolution had taken place, the dosed water (formulation
dissolved in diluent) achieved the target dosed pH of 5.0-
8.0, preferably, 6.0-6.8 and the target dosed ANC of no more
than 100 mg/1 bicarbonate alkalinity, preferably no more than
50 mg/1 bicarbonate alkalinity.
An NaDCC based tablet was prepared containing, along with
various components customarily employed to facilitate
tabletting and to promote dissolution of the tablet by means
of effervescence, a comestible agent present in a
specifically adjusted amount to achieve a desired target pH
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and FAC (free available chlorine) value in the dosed water
when the tablet was completely dissolved in the diluent
water. Dissolution requires that all the tablet components
are uniformly distributed in the diluent, that the solution
is fully diluted to the required dilution for its use as a
microbiocidal agent and that following partial or virtually
complete loss of the excess dissolved carbon dioxide
introduced by the effervescence.
The comestible acid (or a salt thereof) may be a comestible
agent additional to "conventional" tabletting agents and/or
effervescence agents, but the amount of comestible agent must
be specifically determined to attain the desired pH and FAC
value, following loss of excess dissolved carbon dioxide to a
contiguous gaseous phase.
The amount of comestible agent required to produce the
desired pH and FAC value in the dosed water is determined by
a calculation which takes account of the following factors:
(i) The pH of the diluent water;
(ii) The acid neutralising capacity (ANC), classically
referred to as the total alkalinity (as defined by
APHA Standard Methods), of the diluent water;
(iii) The amount of halogen donor such as NaDCC, together
with the amounts of the other dosants of a
formulation, to be added to a unit volume of the
diluent water;
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(iv) The numerical values of the complete set of
equilibrium constants appropriate to the conditions
of the diluent water and defining the equilibrium
conditions of the set of interconverting chemical
5 species derived from the halogen donor, such as
NaDCC, the chemical species derived from the
carbonate and bicarbonate salts, the chemical
species derived from any lubricating agents
(including comestible acids incorporated for that
10 purpose) in the formulation, and the chemical
species derived from the comestible acid which is
to serve as the pH- and FAC-adjusting species as
defined hereinabove;
(v) The pH desired for the dosed water following
complete dissolution of the formulation, following
partial or virtually-complete loss of the excess
carbon dioxide, which is defined as a "final or
ultimate degree of saturation with respect to
carbon dioxide" in paragraph (vii) below;
(vi) The free available halogen donor or chlorine (FAH
or FAC) concentration or, alternatively, the
hypochlorous acid concentration or, alternatively,
the hypochlorite ion concentration, desired for the
dosed water following complete dissolution of the
formulation and loss of excess dissolved carbon
dioxide as described in paragraph (v). The desired
final FAC or hypochlorous acid or hypochlorite ion
concentration may be prescribed [but are not
essentially prescribed] as a fraction of the total
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concentration of chlorine (C1T) introduced into the
diluent water, at its desired dilution, by the
NaDCC component of the formulation (C1T, expressed
as mol per unit volume, is twice the number of mols
of NaDCC added to the unit volume of the diluent).
(vii) The "degree of saturation with respect to carbon
dioxide", referred to in paragraph (v), is the
concentration of free carbon dioxide in a water, in
this case, the dosed and fully diluted water,
expressed as a fraction of the concentration of
free carbon dioxide which would be present if the
water was at equilibrium with a contiguous gaseous
phase for which the partial pressure of carbon
dioxide is known or prescribed;
(viii) The concentration of "free" carbon dioxide,
referred to in paragraph (vii), can be interpreted
as the concentration of C02 per se [as opposed to
the concentration of "dissolved" carbon dioxide
which embraces the dissolved molecular species CO2,
in addition to carbonic acid (HzC03) and its
dissociation products, the bicarbonate (HC03) and
carbonate ions ( C032 ) ] .
(ix) The numerical value of the "degree of saturation
with respect to carbon dioxide", referred to in
paragraph (vii), is obtained by multiplying the
partial pressure of carbon dioxide in the gaseous
phase by the value of the Henry's law constant for
carbon dioxide for the condition of the water and
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for "free" carbon dioxide in accordance with the
alternatives described in paragraph (viii).
Investigation of the impact of total alkalinity or ANC on
microbiocidal efficacy in an effervescent tablet formulation
containing a commonly used active ingredient as a halogen
donor (NaDCC) clearly demonstrates that, when alkalinity is
taken into consideration and an agent to neutralise same is
added to the formulation, there is an immediate improvement
in the microbiocidal efficiency as shown in comparative
trials described in the following examples. These laboratory
trials were carried out with three chlorine donors, as the
chosen halogen, because chlorine is a commonly used halogen
in commercial microbiocides, namely, sodium hypochlorite; a
commercial NaDCC based product, AgriseptR Tabs; and a test
product, the same formulation as AgriseptR Tabs, but
containing, in addition, an agent to neutralise the diluent
alkalinity.
Typically, if the above-mentioned calculation is applied,
then, as two critical factors are varied, so too does the
required quantity of the alkalinity neutralising agent vary.
It has been found that the two factors most likely to be
altered by field conditions are total alkalinity or ANC of
the diluent water; and the volume of the diluent water;
It is clear that, when the ANC increases, then the required
amount of the alkalinity neutralising agent also increases.
However, it should be remembered that, when the volume of
diluent water is increased, the total amount of ANC in the
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water also increases, whereas the amount of alkalinity
neutralising agent in any given tablet formulation is fixed.
By use of a predictive computer programme based on the above-
mentioned factors (i) to (ix), it is possible to calculate
how to vary the quantity of two potential alkalinity
neutralising agents, citric acid and succinic acid, so as to
achieve the target dosed ANC value using a specified volume
of a diluent and using different volumes of a diluent.
Table 1 shows, using this predictive computer programme, the
impact of altering the diluent's ANC, whilst maintaining all
other factors constant. The formulation comprises (excluding
the alkalinity reducing agent) 2.218 NaDCC, 1.138 adipic
acid, 1.0368 sodium bicarbonate and 0.0448 sodium carbonate.
The diluent water pH is 7.5 and its volume is 11. The target
dosed pH and FAC to total C1 ratio are 6.0 and 0.5,
respectively.
Table 1
Diluent Calculated Alkalinity Amount of
Alkalinity Total Carbonic Neutralising Succinic or
(mg/1 as Carbon Agent Citric Acid
calcium (mmol/1) (/tablet) Required
carbonate) (g/tablet)
100 2.1 0.9 0.034
200 4.2 3.7 0.171
400 8.5 9.2 0.445
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Table 2 shows, using the predictive computer programme, the
impact of altering the diluent volume, whilst maintaining all
other factors constant. The formulation comprises (excluding
the alkalinity neutralising agent} 2.218 NaDCC, 1.138 adipic
acid, 1.0368 sodium bicarbonate and 0.0448 sodium carbonate.
The diluent water pH is 7.5 and its volume is 101. The
target dosed pH and FAC to total Cl ratio are 6.0 and 0.5,
respectively.
Table 2
Diluent Calculated Alkalinity Amount of
Alkalinity Total Carbonic Neutralising Succinic or
(mg/1 as Carbon Agent Citric Acid
calcium (mmol/1) (/tablet) Required
carbonate) (g/tablet)
25 0.5 5.1 0.239
100 2.1 22.3 1.267
200 4.2 37.4 2.638
400 8.5 54.9 5.379
Table 3 shows the impact of altering the amount of adipic
acid (the conventional lubricating agent), whilst maintaining
all other factors constant. The composition of the
formulation is as set out above for Tables 1 and 2. The
diluent water volume is 11, its pH is 7.5 and its total
alkalinity is 400 mg/1. The target dosed pH and FAC to total
C1 ratio are 6.0 and 0.5, respectively.
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Table 3
Adipic Acid Amount of Succinic or Total
Succinic Acid Citric Acid Alkalinity
or Citric (/tablet) Reducing
Acid Required Agent
(g/tablet) (/tablet)
g/tablet o/tablet
1.0 20.6 0.561 11.6 32.2
1.25 25.6 0.339 6.9 32.5
1.50 30.6 0.117 2.4 33.0
The impact on microbiocidal efficiency of halogen-based
5 microbiocides, in particular, and, more generally, of any
microbiocide whose activity in solution is influenced or
degraded by the ANC value of the diluent water is as follows:
Table 4
[Microbiocide] Diiuent Volume [ANC] Impact Example
High Medium Low Little 7, 9
Low Low Low Medium
High High Low Medium
Low High Low Major 11
High Medium High Medium 8
Low Low High Major 12
High High High Medium 13
Low High High Major 10
It will be appreciated that the microbiocidal formulation of
the present invention is particularly applicable to
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neutralising the medium and major negative impacts set out
hereinabove and is most particularly applicable to
neutralising the major negative impacts set out hereinabove.
The invention will now be understood in greater detail from
the following description of preferred embodiments thereof
given by way of example only.
A microbiocidal formulation of the present invention has the
following preferred composition:
Ingredient Unit o By Function Reference
Weight Per to
Tablet Standards
Succinic Acid 53.1 Reduces alkalinity EP
Sodium Dichloro- 23.5 Source releasing HSE
isocyanurate hypohalous acid
tanhydrous) into solution
Sodium 11 Carbon dioxide EP
bicarbonate yielding component
of effervescent
base
Adipic Acid 12 Component of HSE
effervescent base
and tabletting
lubricant; reduces
alkalinity
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Ingredient Unit ~ By Function Reference
(contd.) Weight Per (contd.) to
Tablet Standards
(contd. ) (contd. )
Sodium Carbonate 0.5 Component of HSE
(anhydrous) effervescent base
and moisture
stabilising agent
A to aqueous solution of sodium dichloroisocyanurate has a pH
within the range of 5.5-7Ø The incorporation of an
alkalinity reducing agent such as succinic acid ensures that
the alkalinity, which is primarily bicarbonate alkalinity, is
neutralised. This ensures that the nominal amount of the
hypochlorous acid and/or the hypochlorite salt present in the
sodium dichloroisocyanurate solution, is actually available.
The final pH will of course determine the relative amounts of
hypochlorous acid and hypochlorite salt present.
The incorporation of adipic acid ensures a stable formula
suitable for tabletting and the incorporation of the
effervescent excipients ensure the effective and rapid
release of hypochlorous acid and/or hypochlorite salt into
solution, from the source.
It will be appreciated that the identity of the excipients
can be changed or the excipients can be eliminated, depending
on the final requirements or conditions under which the
microbiocidal formulation of the present invention is to be
used.
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The microbiocidal formulation of the present invention will
be equally suitable for use in powder, granulate or tablet
form, each of which allows accurate quantification of the
microbiocidally inactive source and of the excipient
S quantities, in a given volume of water or any other suitable
solvent. The studies set out in the remaining Examples were
carried out with tablets of the microbiocidal formulation of
the invention having the following composition:-
Ingredient Weight (g/tablet)
Succinic Acid 5.00
Sodium dichloroisocyanurate 2.21
Sodium Bicarbonate 1.036
Adipic Acid 1.13
Sodium Carbonate 0.044
Total Tablet Weight 9.42
The alkalinity reducing agent can be integrally incorporated
in the microbiocidal formulation. Alternatively, the
alkalinity reducing agent can be present in a coating on a
powder, granulate or tablet embodiment of the microbiocidal
formulation and, in that event, the alkalinity reducing agent
is released into solution, to reduce the alkalinity level,
before the microbiocidally active agent is released into
solution. It will be appreciated that there is no need for a
tabletting or lubricating agent in a powder or granulate
formulation.
The desired pH of the final solution, in the case of sodium
dichloroisocyanurate as the microbiocidally inactive source,
is in the range 5.0-8.0, preferably 6.0-6.8.
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The dissociation of hypochlorous acid into the hypochlorite
ion is pH dependent. Thus, at pH 6.0, 97.30 of the
hypachlorous acid is undissociated and, at pH 7.0, 78.10 of
the hypochlorous acid is undissociated.
It is preferred that the anhydrous form of the
microbiocidally inactive source be employed in the present
microbiocidal formulations.
Exa~~r~ 1 a 2
The effect of varying water alkalinity on the pH of the
microbiocidal formulation in the environment was investigated
in the following manner:-
Hard water, with a hardness of 342mg/1, is prepared by
dissolving 304mg CaCl2 and 139mg MgC12.6H20 in 1 litre
deionised water. Hard water, with an alkalinity of 100mg/l,
200mg/1 or 300mg/1 is prepared by dissolving 200mg, 400mg or
600mg NaHC03, with 304mg CaClz and 139mg MgC12.6Hz0 in 1 litre
deionised water.
The microbiocidal formulation of the invention comprises one
9.428 tablet of Example 1 dissolved in sufficient hard water
of 0, 100mg/1, 200mg/1 or 300mg/1 added alkalinity, to yield
25 ppm of available chlorine.
The comparative formulation has the following composition:
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Ingredient Weight
(o by weight per tablet)
Sodium dichloroisocyanurate 50
Sodium Bicarbonate 22
Adipic Acid 24
Sodium Carbonate 4
One tablet (5g) of the comparative formulation is dissolved
in sufficient hard water of 0, 100mg/1, 200mg/1 or 300mg/1
5 added alkalinity, to yield 25 ppm available chlorine.
In each case, the pH was measured at 10°C and the results are
shown in Table 5.
Table 5: Effect of Alkalinity on pH
pH
Added Alkalinity Hard Water Comparative Formulation
(ppm) (Diluent) Formulation of Invention
(Dosed Water) (Dosed Water)
0 7.66 7.71 6.05
100 7.51 7.70 6.60
200 7.93 7.72 6.70
300 8.08 7.78 6.72
An impact of alkalinity on the dissociation of hypochlorous
acid into hypochlorite and, therefore, on microbiocidal
efficacy is suggested by the above-mentioned results. Thus,
current experimental data generated in standard hard water
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are likely to be inaccurate in field conditions where
alkalinity is often present.
It is clear that added alkalinity affects the pH of both the
comparative formulation solution and the formulation of the
invention solution. Specifically, when the added alkalinity
is greater than 100mg/1, the pH of the comparative
formulation solution is outside the desired 6.0-6.8 pH range.
In contrast, even when the added alkalinity is 300mg/1, the
pH of the formulation of the invention solution is still
within the desired 6.0-6.8 pH range.
The usual recommended method of carrying out the cleaning and
disinfection of an automated milking system is known as cold
circulation cleaning.
By this procedure, the lumen of the milking system is pre-
rinsed at the end of the morning milking process, with cold
water, followed by a wash sequence involving circulation of
45 litres of a detergent solution containing 0.5% (227g/45
litres (0.51b/10ga1)) of an approved caustic detergent, for
10 minutes, which detergent solution is then recovered in the
wash trough for reuse in the second daily wash. It is
suggested not to post-rinse the caustic solution residue from
the lumen until immediately before the next milking, since it
is believed that successful cleaning and microbiocidal action
on the lumen of the milking system depends on prolonged
contact of this caustic residue with the lumen surfaces. A
hot wash at regular intervals is an essential part of the
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routine to remove built up milk deposits, inter alia. In
order to get optimum results from cold cleaning, the
following instructions are widely acceptable:-
Prescribed Routines:
A) Cold Circulation Cleaning
Wash fetters and outside of clusters and attach them to the
fetters. Rinse lumen of system with 14 litres (3 gal) cold
water per cluster. Dissolve an approved caustic detergent in
cold water at the rate of 227g/45 litres (0.51b/lOgal)
allowing about 9 litres (2 gal) of solution per cluster.
Circulate the solution for 10 min having allowed the first 5
litres (1 gal) to run to waste. Return all the solution to
the wash trough and retain for the second daily wash. Leave
the clusters on fetters. Before next milking, rinse lumen of
system with 14 litres (3 gal)~cold water per cluster to
remove the caustic detergent residue. Add 28m1 (1 fl. oz)
agricultural grade hypochlorite to the final ~7 litres (10
gal) of rinse water.
B) Regular hot wash (recommended at fortnightly intervals).
The data set out in Table 6 were obtained from two milking
parlours on the same farm, one of which (control parlour)
used the above-mentioned prescribed routine and the other of
which (trial parlour) used the following alternative routine.
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Wash fetters and outside of clusters and attach them to the
fetters. Rinse the milking system through with 14 litres of
cold clean water per cluster. Dissolve an approved caustic
detergent in cold water at the rate of 2278/45 litres
(0.51b/lOgal) allowing about 9 litres (2 gal) of solution per
cluster. Make this cold wash up each day and use twice only.
Having allowed the first 5 litres (1 gal) to run to waste,
circulate the solution for 10 minutes. Return all the
remaining solution to the wash trough and retain for the
second daily wash. Leave clusters on fetters. Next, post-
rinse (at 2pm after morning use s2~ immediately after evening
use) the system with 14 litres (3 gal) of cold clean water
per cluster, to remove all traces of the caustic detergent
residue. Prepare a microbiocidal formulation of the
invention (dissolve two 9.428 tablets in about 11 water; the
tablet comprises 5.Og succinic acid, 2.218 sodium
dichloroisocyanurate, 1.0368 sodium bicarbonate, 1.138 adipic
acid and 0.0448 sodium carbonate) and add same to 661 of the
rinse water of the final rinse cycle. Suck through the
milking system and allow to drain completely.
The trial was continued for two weeks. Table 6 gives the
week 1 and week 2 results of the microbiological count of
plant rinses after circulation during the trial.
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TABLE 6: Microbiological count of plant rinses (count per ml
of rinse water)
Microbiological Week 1 Week 2
Count Control Trial Control Trial
Total bacterial 1300 380 600 410
Count
Psychotrophic 340 119 80 5
Thermoduric 3 1 4 5
The total bacterial counts and the psychotrophic counts for
each of week 1 and week 2 show a superior performance for the
microbiocidal formulation of the present invention. In
addition, there was little evidence of a protein-like film
build-up after two weeks use of the microbiocidal formulation
of the invention.
A laboratory comparison was carried out to determine the
microbiocidal efficacy of the test formulation as described
in Example 1, of the comparative formulation (5g tablets) as
described in Example 2 and of a sodium hypochlorite solution
(Merck), supplied by Lennox Chemicals, Dublin, Ireland. The
objective was to establish that the formulation of the
present invention was more effective as a microbiocide than a
conventional sodium hypochlorite solution and a conventional
tableted formulation, as per Example 2, in "field" water.
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The general format of the testing schedule is derived from
BS3286:1960. The tests were carried out using 0.1% milk as
an organic load. The milk used was unpasteurised milk with
an approximate somatic cell count of 300 x 103. The test
5 organisms were Staphylococcus aureus, isolated from a
clinical case of mastitis in dairy cows, and Bacillus
subtilis, a control culture obtained from ATCC. Contact
times for the three products were 5 mins, 10 mins, 6 hrs and
24 hrs .
The concentrations of the three products were 25 ppm (mg/1)
of available chlorine. The tests were carried out at 10°C.
Dilutions in all cases were made in a diluent comprising
water with an alkalinity of 300 mg/1 expressed as calcium
carbonate (CaC03) equivalent and a hardness of 342mg/1
expressed as calcium carbonate (CaC03). The pH of the water
was checked before and after addition of trial product.
Inactivation of the products under test was carried out by
placing lml of reactant mix, after the appropriate contact
time, in 9mls of sterile inactivation fluid. The
inactivation will neutralise the effect of the disinfectant.
Dilutions were made at the contact times mentioned, and the
inactivated fluids were cultured onto blood agar and
MacConkey agar using standard laboratory practices.
Incubation was carried out for 18-24 hrs at 37°C. The
inoculum consisted of 6mls of the test organism (1 x 104
orgs/ml) plus 4 mls of O.lo milk. The disinfectant
dilution:inoculum ratio was 50:50. Controls substituted
water, i.e. water with a hardness of 342mg/1 and an
alkalinity of 300mg/1, instead of the trial product. The
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minimum level (pass criteria) that is required will be 99.99%
over viable count (cfu/ml).
The inactivation fluid comprises lecithin - soya (3g), Tween
80 (30m1), sodium thiosulphate (5g), L-histidine (lg),
phosphate buffer (0.25N; 10m1), and purified water - made up
to 1 litre. The inactivation fluid was sterilised at 121°C
for 15 minutes.
The "field" diluent comprises NaHC03 (600mg), CaClz (304mg)
and MgC12.6Hz0 (1339mg), which is made up to 1,OOOml with
deionised water.
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RESULTS
Table 7
pH of "field"diluent . 7.98
pH of 25 ppm sodium hypochlorite solution . 8.21
pH of 25 ppm Agrisept Tabs solution . 7.73
pH of 25 ppm formulation of invention solution . 6.80
10
Organism: Bacillus subtilis
Product (cfu)
Time Sodium Agrisept Formulation of
Hypochlorite Invention
5 mins >100 cfu* >100 cfu 59
mins >100 cfu >100 cfu 90
6 hrs >100 cfu >100 cfu 18
24 hrs >100 cfu 8 cfu N/G
N/G = No Growtri ~onLrol ~.vu~~~
*The indication of >100 cfu/ml means that the organisms were
too numerous to quantify.
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Organism: Staphylococcus aureus
Product
Time Sodium Agrisept~ Trial Product
Hypochlorite
mins >100 cfu N/G N/G
20 mins 88 cfu N/G N/G
6 hrs N/G N/G N/G
24 hrs N/G N/G N/G
5 N/G = No Growth Control Count 1 x 10°
A laboratory comparison of the efficiency of a test
formulation as described in Example 1 against the
conventional microbiocide, sodium hypochlorite, at two
different concentrations, under control conditions. The
objective was to establish that the formulation of the
present invention was more effective as a microbiocide at a
very low concentration, 25 ppm, when compared to sodium
hypochlorite and that, even if the concentration was
increased, by a factor of 10, to 250 ppm, the formulation of
the present invention was much more efficient in "field"
water.
The testing procedure of Example 4 was followed, with the
following amendments. The test organisms were Bacillus
subtilis, Salmonella typhirnurium (phage type 104), Listeria
monocytogenes and Clostridium butyricum.
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The concentrations were 25 ppm and 250 ppm for each of
hypochlorite and formulation of the present invention. The
organic load was 0.1% milk (25 ppm) or 5o yeast (250 ppm).
TABLE 10
Organism: Salmonella typhimurium phage type 104
25 ppm 250 ppm
Time Control Test Control Test
(Hypochlorite) Product (Hypochlorite) Product
5 mins 200-500 100-200 100-200 0
mins 100-200 100-200 100 0
30 mins 100-200 100 100-200 0
1 hour 100-200 100-200 100-200 0
6 hours 100-200 42 100 0
24 hours 100-200 100-200 100 0
TABLE 11
10 Organism: Listeria monocytogenes
25 ppm 250 ppm
Time Control Test Control Test
(Hypochlorite) Product (Hypochlorite) Product
5 mins 100-200 100-200 91 0
10 mins 100-200 100-200 200-500 0
30 mins 100-500 100-200 200-500 0
1 hour 200-500 100-200 200-500 0
6 hours 100-200 100-200 100-200 0
24 hours 100-200 0 >200 100-200
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TABLE 12
Organism: Clostridium butyricum
25 ppm 250 ppm
Time Control Test Control Test
(Hypochlorite) Product (Hypochlorite) Product
5 mins 66 20 63 10
10 mins 48 32 41 5
30 mins 34 26 61 6
1 hour 33 9 40 3
6 hours 13 18 13 4
24 hours 0 0 0 0
5 TABLE 13
Organism: Bacillus subtilis
25 ppm 250 ppm
Time Control Test Control Test
(Hypochlorite) Product (Hypochlorite) Product
5 mins 28 23 27 10
10 mins 31 28 26 13
30 mins 21 23 35 4
1 hour 21 12 28 4
6 hours 26 32 23 1
24 hours 15 28 22 0
These data demonstrate the difference between sodium
10 hypochlorite and the formulation of the present invention.
The trial was carried out simultaneously on both products at
two different concentrations, 25 ppm and 250 ppm. The
results of the trial clearly demonstrate that, at 250 ppm,
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the formulation of the present invention is microbiocidally
superior to the conventional hypochlorite. At 25 ppm, the
difference is smaller but, in most cases, the formulation of
the present invention is still superior.
Formulation (A) Total Formulated Weight.....3.2 grams
Composition: Sod. Dichloroisocyanurate... 25% (w/w).....800mg
Sod. Bicarbonate............ 350 (w/w)....1120mg
Sod. Carbonate.............. .5% (w/w).....160mg
Succinic Acid...............35% (w/w)....1120mg
Formulation (B) Total Formulated Weight.....5.56 grams
Composition: Sod. Dichloroisocyanurate...14.39% (w/w)...800mg
Sod. Bicarbonate............20.14% (w/w)..1120mg
Sod. Carbonate..............-2.880 (w/w)...160mg
Succinic Acid...............62.59% (w/w)..3480mg
The general format of the testing schedule is derived from
BS3286:1960 and the testing procedure of Example 4 was
followed, with the following amendments. The tests were
carried out using 0.1% milk as an organic load. The test
organism was Bacillus subtilis BGA, a control culture
obtained from ATCC. The contact time for each of the
formulations was 5 mins, 10 mins, 1 hour and 6 hours. The
concentration of each formulation was 100 ppm (mg/1)
available chlorine in a volume of 5 litres of diluent water.
All tests were carried out at 10°C. The diluent water had a
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total alkalinity of 400mg/1 expressed as calcium carbonate
(CaC03) equivalent and a hardness of 342mg/1 expressed as
calcium carbonate (CaC03). The pH of the water was measured
before and after the addition of the various formulations.
The inactivated fluids were cultured onto Colombia Blood Agar
using standard laboratory practices. Incubation was for 18
hours at 37°C. The concentration of the inoculum was 1.25 x
106 orgs/ml. The "field" diluent comprises NaHC03 (750mg),
CaCl2 (304mg) and MgC12.6Hz0 (1339mg), which is made up to
1,OOOm1 with deionised water.
Table 14
pH of "field" diluent: 8.01
pH of 100 ppm solution of test formulation A (3.2g): 6.68
pH of 100 ppm solution of test formulation B (5.52g): 5.41
Table 15
Contact Time Test Formulation A Test Formulation B
5 mins >100 cfu* 89
10 mins >100 cfu 52
1 hour >100 cfu 64
6 hours >100 cfu 91
*The indication of >100 cfu/ml means that the organisms were
too numerous to quantify.
The results show that Test Formulation B is superior to Test
Formulation A in suppressing growth of Bacillus subtilis BGA
under the conditions described. The residual resistance of
the organism is due to various factors limiting the
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formulation's microbiocidal activity, specifically, its spore
form, the high concentration of the inoculum organism used
and the high total alkalinity of the diluent.
It will be appreciated that the microbiocidal formulation of
the present invention has applicability in a milking
apparatus at the end of a post-milking apparatus washing
procedure (as exemplified in Example 3); in any pre-
pasteurisation holding system; and in any post-pasteurisation
apparatus against post-pasteurisation contaminant micro-
organisms.
Although the above-mentioned results concern sterilisation of
the lumen of a milking apparatus, it is expected that the
microbiocidal formulations of the present invention will be
particularly suited to the treatment of water, where the
world Health Organisation (WHO) have now set the limit for
residual chlorine in treated water at only 5mg/1. Thus, with
conventional microbiocidal formulations, such water is being
inadequately treated, since at least some of the hypohalous
acid/hypohalite salt is being neutralised by the alkalinity
present and, if excess conventional microbiocidal formulation
is provided, then, in areas of low alkalinity, the WHO limits
may be exceeded.
It will be appreciated, therefore, that the microbiocidal
formulations of the present invention provide effective water
treatment by giving effective microbiocidal effect without
exceeding the WHO recommended limits.
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It will also be appreciated that the microbiocidal
formulation of the present invention, although exemplified in
respect of a milking apparatus, has general applicability in
any apparatus used in the production, preparation or
processing of food or beverages and, indeed, in the treatment
of water for human or animal consumption or of process
liquids.
Sodiumdichloroisocyanurate - 2.2108
Adipicacid - 1.1308
Sodiumbicarbonate - 1.0368
Sodiumcarbonate - 0.0448
Succinic - 0.5618
acid
A microbiocidal formulation of the above-mentioned
composition is useful, following dissolution in five litres
of a diluent having a pH of 7.5, a total alkalinity of
100mg/1 as calcium carbonate and a total carbonic carbon of
2.lmmol/1 (calculated) so as to achieve, following
dissolution in the diluent, a pH of 6.0 and a FAC to total Cl
ratio of 1Ø
2 0 ~x~ l~e-8
Sodiumdichloroisocyanurate - 2.2108
Adipicacid - 1.1308
Sodiumbicarbonate - 1.0368
Sodiumcarbonate - 0.0448
Succinic - 2.6198
acid
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A microbiocidal formulation of the above-mentioned
composition is useful, following dissolution in five litres
of a diluent having a pH of 7.5, a total alkalinity of
400mg/1 as calcium carbonate and a total carbonic carbon of
5 8.5mmo1/1 (calculated) so as to achieve, following
dissolution in the diluent, a pH of 6.0 and a FAC to total C1
ratio of 1Ø
Sodium dichloroisocyanurate - 2.210g
Adipic acid - 1.1308
Sodium bicarbonate - 1.0368
Sodium carbonate - 0.0448
Succinic - 0.2188
acid
A microbiocidal formulation of the above-mentioned
composition is useful, following dissolution in five litres
of a diluent having a pH of 7.5, a total alkalinity of 50mg/1
as calcium carbonate and a total carbonic carbon of l.lmmol/1
(calculated) so as to achieve, following dissolution in the
diluent, a pH of 6.0 and a FAC to total C1 ratio of 1Ø
Sodium dichloroisocyanurate - 2.2108
Adipic acid - 1.1308
Sodium bicarbonate - 1.0368
Sodium carbonate - 0.0448
Succinic - 13.5898
acid
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A microbiocidal formulation of the above-mentioned
composition is useful, following dissolution in twenty-five
litres of a diluent having a pH of 7.5, a total alkalinity of
400mg/1 as calcium carbonate and a total carbonic carbon of
8.5mmol/1 (calculated) so as to achieve, following
dissolution in the diluent, a pH of 6.0 and a FAC to total C1
ratio of 1Ø
Sodiumdichloroisocyanurate - 2.210g
Adipicacid - 1.130g
Sodiumbicarbonate - 1.0368
Sodiumcarbonate - 0.0448
Succinic - 3.3038
acid
A microbiocidal formulation of the above-mentioned
composition is useful, following dissolution in twenty-five
litres of a diluent having a pH of 7.5, a total alkalinity of
100mg/1 as calcium carbonate and a total carbonic carbon of
2.lmmol/1 (calculated) so as to achieve, following
dissolution in the diluent, a pH of 6.0 and a FAC to total Cl
ratio of 1Ø
Ex~~le 12
Sodiumdichloroisocyanurate - 2.2108
Adipicacid - 1.1308
Sodiumbicarbonate - 1.0368
Sodiumcarbonate - 0.0448
Succinic - 0.4288
acid
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A microbiocidal formulation of the above-mentioned
composition is useful, following dissolution in one litres of
a diluent having a pH of 7.5, a total alkalinity of 400mg/1
as calcium carbonate and a total carbonic carbon of 8.5mmo1/1
(calculated) so as to achieve, following dissolution in the
diluent, a pH of 6.0 and a FAC to total C1 ratio of 1Ø
Sodium dichloroisocyanurate - 2.2108
Adipic acid - 1.1308
Sodium bicarbonate - 1.0368
Sodium carbonate - 0.0448
Succinic - 0.0178
acid
A microbiocidal formulation of the above-mentioned
composition is useful, following dissolution in one litres of
a diluent having a pH of 7.5, a total alkalinity of 100mg/1
as calcium carbonate and a total carbonic carbon of 2.lmmol/1
(calculated) so as to achieve, following dissolution in the
diluent, a pH of 6.0 and a FAC to total C1 ratio of 1Ø
Referring to Examples 7-13, Example 13 is an example of a
microbiocidal formulation, in which the total alkalinity of
the diluent water plays a very minor role, in that it is
necessary to merely add 0.0178 succinic acid to the adipic
acid already present in the microbiocidal formulation, in
order to achieve a target dosed pH of 6Ø
Concerning Examples 7, 9 and 12, the total alkalinity of the
diluent water plays a minor role, in that it is necessary to
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add 0.2-0.6, preferably, 0.218-0.5618 succinic acid to the
adipic acid already present in the microbiocidal formulation,
in order to achieve a target dosed pH of 6Ø
Concerning Example 8, the total alkalinity of the diluent
water plays a medium role, in that it is necessary to add
2.6198 succinic acid to the adipic acid already present in
the microbiocidal formulation, in order to achieve a target
dosed pH of 6Ø
Concerning Examples 10 and 11, the total alkalinity of the
diluent water plays a major role, in that it is necessary to
add 3-158, preferably, 3.303-13.5898 succinic acid to the
adipic acid already present in the microbiocidal formulation,
in order to achieve a target dosed pH of 6Ø
